Project Summary/Abstract: Human skin and mucosa are composed predominantly of keratinocytes, which play a critical role in maintaining organ and organismal barriers against environmental insults. Included in such insults are endogenous and exogenous DNA toxins that cause genome instability and DNA damage. DNA repair mechanisms are a critical secondary defense once DNA damage has occurred. While all cells in the body have evolved sophisticated mechanisms to repair DNA damage, the consequences of defective repair are distinct for different organs and are likely rooted in developmental processes that remain poorly understood. Nowhere is the importance of organ-specific DNA repair more apparent than in the inherited human genome instability disorder Fanconi Anemia (FA), wherein every cell in the body is incapable of repairing certain types of DNA damage properly. A universal molecular feature of the disease is the absence of the FA DNA repair pathway. Clinical features of the disease include congenital abnormalities, bone marrow failure and, curiously, a unique susceptibility to keratinocyte transformation. FA patients exhibit an extreme susceptibility to squamous cell carcinomas (SCCs) of the skin and mucosa early in life, and we have recently uncovered novel adhesion defects in normal-appearing patient skin using electron microscopic studies. The role of the FA pathway in normal keratinocytes and developmental origins for defective tissue homeostasis and SCC predisposition will be examined here. I hypothesize, based on preliminary data, that FA pathway loss impairs signaling and barrier functions of somatic epidermal stem and progenitor cells (ESPCs) and that these abnormalities, in turn, promote tissue defects in 3D engineered models of skin as well as ultimately SCC susceptibility and progression. These hypotheses will be tested using a novel developmental system wherein induced pluripotent stem cells generated from FA patients are inducibly complemented to reconstitute FA pathway function and differentiated into keratinocytes and 3D epidermis. All experimental elements of the projects are in place and I anticipate a learning experience which will allow me to gain a comprehensive understanding of developmental processes leading to defective somatic ESPCs and resulting inherited disease susceptibilities as a solid academic career foundation.